Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 598313, 11 pages http://dx.doi.org/10.1155/2014/598313

Research Article Genetic Diversity in Assessed by Morphological and ITS Sequence Analysis

Shiamala Devi Ramaiya,1 Japar Sidik Bujang,1 and Muta Harah Zakaria2

1 Department of Animal Science and Fisheries, Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia Bintulu Sarawak Campus, 97008 Bintulu, Sarawak, Malaysia 2 Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Shiamala Devi Ramaiya; shiamala [email protected]

Received 16 January 2014; Revised 13 May 2014; Accepted 15 May 2014; Published 22 June 2014

Academic Editor: Andrea Zuccolo

Copyright © 2014 Shiamala Devi Ramaiya et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This study used morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA to investigate the phylogeny of Passiflora species. The samples were collected from various regions of East Malaysia, and discriminant function analysis based on linear combinations of morphological variables was used to classify the Passiflora species. The biplots generated five distinct groups discriminated by morphological variables. The group consisted of of P. edu li s with high levels of genetic similarity; in contrast, P. fo eti d a was highly divergent from other species in the morphological biplots. The final dataset of aligned sequences from nine studied Passiflora accessions and 30 other individuals obtained from GenBank database (NCBI) yielded one most parsimonious with two strongly supported clades. Maximum parsimony (MP) tree showed the phylogenetic relationships within this subgenus Passiflora support the classification at the series level. The constructed phylogenic tree also confirmed the divergence of P. fo eti d a from all other species and the closeness of wild and cultivated species. The phylogenetic relationships were consistent with results of rphologicalmo assessments. The results of this study indicate that ITS region analysis represents a useful tool for evaluating genetic diversity in Passiflora at the species level.

1. Introduction undergone significant revision based on morphological traits by Feuillet and MacDougal [7] and four subgenera were rec- Passiflora is extensively grown in tropical and subtropical ognized: Passiflora, Decaloba, Deidamioides, and Astrophea. regions of the world. More than 500 species in this genus have While progress has been made in understanding phylogenetic been identified, and most of them are distributed throughout relationships of subgenus Passiflora, little is known about Central and [1]. is one of the main the relationship among them. The subgenus Passiflora is the centers of Passiflora diversity which accommodates more largest in genus of Passiflora which comprised about 240 than 100 species and is rich in nearly all sections of the species generally regarded as typical passionflowers. Species genus [2]. Passiflora species have been cultivated for their of subgenus Passiflora are characterized by handsome edible , ornamental flowers, and pharmaceutical uses. that are dominated by corona [1]. Feuillet and MacDougal The Passiflora genus contains the highest number of species [7] subdivide the representatives of subgenus Passiflora into in with edible fruit3 [ ]; in addition, the six supersections and further divided into sections or series of this genus have desirable organoleptic properties [4]. based on their morphological characteristics. At the early 20th century, two major technical mono- The species of the Passiflora genus have a wide range graphs laid the modern foundation for Passiflora systematics, of morphological characteristics, anatomical differences, and including works by Harms [5]andKillip[6]. The species were phylogenetic variability. According to Sanchez´ et al. [8], divided into 22 subgenera based on floral morphology by Passiflora species are difficult to classify because some species Killip [6]. Later, the infrageneric of Passiflora has vary widely in terms of morphology and other species closely 2 The Scientific World Journal resemble each other. Taxonomic classifications of Passiflora subgenus Passiflora and DNA studies using sequence data aretypicallybasedonmorphological[9–11], ecological [12], from the genome may more accurately define this phylogeny. and agronomic variations [13]. For example, P. e du li s is Accessing molecular polymorphisms at the species level also generally characterized according to production, weight, contributes to the acquisition of knowledge that can be fruit size, juiciness, and juice acidity; however, these variables useful for the conservation of the diversity of Passiflora. do not represent precise quantitative traits at the taxonomic Interest in this agronomically important crop (particularly level [9]. Moreover, existing inter- and intraspecies dissimi- in the Passiflora subgenus) has grown. Therefore, we studied larity among the Passiflora species makes understanding the morphological characteristics and phylogenetic relationships link between morphological plasticity, genotypic diversity, of species of this genus by evaluating the genetic diversity andspeciationchallenging.Increasingnumbersofvarious and comparing the ITS sequence data from plants of different interspecific hybrids have been produced in this genus14 [ ], origins. The knowledge generated by these genetic-based thus contributing to broad morphological variability. characterizations provides information on the parental selec- Germplasm characterization based on molecular phy- tion for breeding works and also may contribute to various logeny can also contribute to a better understanding of the aspects of future biological and ecological studies. evolutionary process and genetic divergence of accessions. Assessing genetic diversity in Passiflora is also important for the utilization and conservation of the germplasm. The 2. Materials and Methods genetic background of the is a crucial factor for plant 2.1. Sample Collection and Morphological Analysis. Plant breeders when selecting the parental material for breeding materials were obtained from various regions of East [15, 16]. Over the years, studies have been carried out to Malaysia as presented in Table 1.Plantpartsfromcultivated examine the phylogenetic relationships within the genera. Passiflora accessions P. e du li s producing purple fruits (PE1), Studies have reported attempts to clearly demarcate the P. e du li s producing dark purple fruits (PE2), P. incar nata , P. species of the Passiflora genus using restriction enzymes quadrangularis and P. malifor mi s were collected from the fruit (cpDNA sites; Yockteng and Nadot [17]; Paikrao et al. [18]), farm of Universiti Putra Malaysia Bintulu Sarawak Campus amplified fragments length polymorphism markers (AFLP; ∘ 󸀠 ∘ 󸀠 (UPMKB) Bintulu (N 03 12.45 and E 113 4.68 ), Sarawak. Ortiz et al. [4]; Segura et al. [15]), microsatellite markers The five plant species were grown from acquired from (SSR; Ortiz et al. [4]; Oliveira et al. [19]), and inter-simple the commercial supplier Trade Winds Fruit, Windsor. In sequence repeats (ISSR; dos Santos et al. [11]).However,the addition, P. e du li s producing pink red fruits (PE3) and P. genetic diversity of Passiflora species is mostly estimated with edulis producing yellow fruits (PE4) were collected from random amplified polymorphic DNA (RAPD; Fajardo et al. ∘ 󸀠 small-scale passion fruit farms at Kota Kinabalu (N 05 58.28 [20]; Aukar et al. [21]; Crochemore et al. [22]; Cerqueira-Silva ∘ 󸀠 ∘ 󸀠 and E 116 5.72 ), Sabah and Ba’kelalan (N 03 58.44 and et al. [23]). ∘ 󸀠 E 115 37.08 ), Sarawak, respectively. Wild P. fo eti d a possessing The internal transcribed spacer (ITS) region of the flowers with green bracts (PF1) and P. fo eti d a possessing nuclear ribosomal 18S-5.8S-25S rDNA locus, which has been flowers with red bracts (PF2) were collected from bushes used in phylogenetic studies of many angiosperm families, ∘ 󸀠 ∘ 󸀠 at Bintulu (N 03 10.25 and E 113 2.39 ), Sarawak. Data has proven particularly useful for improving our under- on vegetative and reproductive morphology were recorded, standing of interspecies relationships [24]. The two internal and quantitative features were determined for the aril parts; transcribed spacer DNA sequences have evolved rapidly and , stems, tendrils, bracts, flowers, fruits and seeds. are therefore useful for comparing closely related taxa. In Specimen identification and botanical nomenclature were some genera, variations in ITS sequences have proven useful based on the taxonomic keys of Ulmer and MacDougal [1]. for studies at the species level [25]. The ITS region is also flanked by well-conserved rRNA genes that can be used to differentiate plant species [26]suchaslentils[27], peanuts 2.2. Phylogenetic Analysis [28], maize [29], and seagrass [30]. The ITS regions have also been used to assess the phylogeny of the genus Passiflora. 2.2.1. Polymerase Chain Reaction (PCR) Amplification. DNA In particular, a recent study by Mader¨ et al. [31]used was isolated from 0.01 g of fresh leaves using a modified alka- ITS sequences to evaluate the intraspecific variability of 23 line lysis method without NaOH [35]. The nuclear ribosomal species of Passiflora. The first molecular phylogenetic analysis ITS regions were selected for PCR amplification and sequence of Passiflora using plastid regions and ITS sequences was analysis (forward (ITS1: TCCGTAGGTGAACCTGCGG) conducted by Muschner et al. [32]; these investigators studied and reverse (ITS4: TCCTCCGCTTATTGATATGA)). The the 61 species composing the entire subgenera and identified primers were chosen based on ITS sequences published three major clades (Passiflora, Decaloba, and Astrophea). by White et al. [36]andpurchasedinalyophilizedform Although insights into Passiflora phylogeny at the sub- from First BASE Laboratories, Malaysia. Each PCR reaction generic level have been gleaned, genetic information on and contained 13.0 𝜇L of sterile ultrapure water, 2.0 𝜇Lof10xNH4 evidence for monophyletic groups below this level are limited PCRbuffer,0.4𝜇Lof10𝜇M dNTPs, 2.0 𝜇Lof50𝜇M MgCl2, [15, 33, 34]. At lower levels, the use of an arbitrary selection of 1.0 𝜇Lof10𝜇M forward and reverse primers, 1 unit DNA Taq morphological characteristics to delimit genera has yielded polymerase, and 20–40 ng of template genomic DNA f8 or conflicting results. More information is needed to address a final volume of 20.0 𝜇L. The PCR amplification of the ITS evolutionary questions at the interspecies relationship in region was performed using an XP Thermal Cycler under The Scientific World Journal 3

Table 1: List of Passiflora accessions examined and individuals included in molecular analysis with their geographical locations, GenBank accession numbers, and references. Passiflora accessions Geographical locations GenBank accession number Citation (PE1) Bintulu, Sarawak, and Malaysia Present sequence Present study P. edu li s (PE2) Bintulu, Sarawak, and Malaysia Present sequence Present study P. edu li s (PE3) Kota Kinabalu, Sabah, and Malaysia Present sequence Present study P. edu li s (PE4) Ba’kelalan, Sarawak, and Malaysia Present sequence Present study P. qu adrang u l ar i s Bintulu, Sarawak, and Malaysia Present sequence Present study P. incar nata Bintulu, Sarawak, and Malaysia Present sequence Present study P. malifor mi s Bintulu, Sarawak, and Malaysia Present sequence Present study P. fo eti d a (PF1) Bintulu, Sarawak, and Malaysia Present sequence Present study P. fo eti d a (PF2) Bintulu, Sarawak, and Malaysia Present sequence Present study P. edu li s Brazilian state1,2,3 EU258382.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258381.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258378.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258383.1 Mader¨ et al. [31] P. edu li s Netherland AF454803.1 Ossowski [45] P. edu li s Brazilian state1,2,3 EU258379.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258384.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258380.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258375.1 Mader¨ et al. [31] P. edu li s Brazilian state1,2,3 EU258376.1 Mader¨ et al. [31] P. qu adrang u l ar i s French Guyana AF454799.1 Ossowski [45] P. qu adrang u l ar i s Ohio state AY636107.1 Krosnick and Freudenstein [46] P. al ata Viamao, RS AY032826.1 Muschner et al. [32] P. incar nata Brazilian state DQ344630.1 Muschner et al. [14] P. v itifoli a Unknown AF454796.1 Ossowski [45] P. cae r u l ea Netherland AF454802.1 Ossowski [45] P. cae r u l ea Brazilian state1 EU258315.1 Mader¨ et al. [31] P. ambig u a Unknown AF454801.1 Ossowski [45] P. pl at y l ob a Horticultural, USA AF454798.1 Ossowski [45] P. malifor mi s Dominica AY210956.1 Muschner et al. [32] P. fo eti d a Brazilian state1,2,4 EU258389.1 Mader¨ et al. [31] P. fo eti d a Brazilian state1,2,4 EU258393.1 Mader¨ et al. [31] P. p alme r i Brazilian state1,2,4 DQ238784.1 Muschner et al. [14] P. fo eti d a United States DQ521376.1 Hearn [47] P. fo eti d a Brazilian state DQ238783.1 Muschner et al. [14] P. fo eti d a JQ723359.1 Thulin et al. [48] P. fo eti d a Brazilian state1,2,4 EU258394.1 Mader¨ et al. [31] P. fo eti d a Tropical regions DQ499117.1 Wright et al. [49] ∗ 𝑀𝑖𝑡𝑜𝑠𝑡𝑒𝑚𝑚𝑎 𝑏𝑟𝑒𝑣𝑖𝑓𝑖𝑙𝑖𝑠 Campo Grande5 AY102359.1 Muschner et al. [32] ∗ 𝑃𝑎𝑟𝑜𝑝𝑠𝑖𝑎 𝑚𝑎𝑑𝑎𝑔𝑎𝑠𝑐𝑎𝑟𝑖𝑒𝑛𝑠𝑖𝑠 M Zyhra 949, WIS AY102365.1 Muschner et al. [32] Samples collected from various locations of Brazilian states: RS1: Rio Grande do Sul, SC2: Santa Catarina, MG3:MinasGerais,PB4:Pernambuco,andMS5: ∗ Mato Grosso do Sul; tropical samples collected from New Guinea, northeast Australia, Borneo, India, Tahiti, and South America. Outgroups species. the following conditions: one cycle of initial denaturation with6xloadingbufferandusedtomeasurethesizeofthe ∘ ∘ for3minat95C and 35 cycles of denaturation at 94 Cfor obtained DNA fragments. Gel electrophoresis was run for ∘ 30 s, annealing at 55 C for 30 s, and elongation for 1 min at 90minat90Vusingagelelectrophoresissystem.Thegelwas ∘ ∘ 72 C. The final elongation step was conducted at 72 Cfor photographed using the AlphaView UV light imaging system. 5min.ThePCRproductswerequantifiedusing1%agarosegel The excess dNTPs were removed from the amplified PCR electrophoresis; 6 𝜇LofPCRproductwerepremixedwith6x products using the polyethylene glycol precipitation method loading buffer and loaded with 1 𝜇L of EZ-Vision Fluorescent [37],andthepurifiedPCRproductsweresenttoFirstBase Dye for the visualization of DNA bands in the agarose Laboratory, Malaysia, for DNA sequencing. Both strands gel. The GeneRuler 1 kb plus DNA ladder was premixed of the PCR product (reverse and forward) were sequenced 4 The Scientific World Journal using the Applied Biosystems BigDye Terminator v3.1 cycle models option used in other models, MEGA uses MP search sequencing kit. model option to implement parsimony. The basic premise of parsimony is that taxa share a common characteristic because they inherited that characteristic from common ancestors 2.3. Statistical Analysis. Data on morphological variables [41]. We used the heuristic search method with simple taxon ( length, leaf width, stem width, tendril length, tendril addition and tree bisection reconnection (TBR). The choice width, bract length, bracts width, size, petal length, of nodes for branch swapping in the resulting parsimonious petal width, sepal length, sepal width, number of outer rows model was informed by bootstrap analyses consisting of 1000 of corona, corona length, fruit weight, fruit length, fruit replications of the heuristic search. The MEGA 5.1 program width, length, and seed width) were statistically analyzed was also used to construct the phylogenetic tree and estimate using the SAS 9.0 for Windows. Single-factor analysis of sequence divergence [40]. variance (ANOVA) with post hoc Tukey’s test (𝑃 ≤ 0.05)was used to compare the mean values. Discriminant analysis (DA) basedonlinearcombinationsofthepredictorvariableswas 3. Results and Discussion used to find the maximum separation between the studied Passiflora species using XLSTAT 2013 for Windows. 3.1. Morphological Variation and Discriminant Analysis (DA). The electropherograms of the sequence fragments were Variations in the nineteen studied morphological character- inspected and assembled using Phred, Phrap, and Consed istics of the Passiflora species are presented in Table 2.The softwareinMacPro[38]. The chromatograms were ana- vegetative and reproductive data were analyzed for discrimi- lyzed with Phred, assembled with Phrap, and scanned with nant analysis. Discriminant function analysis based on linear PolyPhred; the results were then viewed with the Consed pro- combinations of the variables produced better discrimination gram. Only good-quality fragments (sequence quality above of the Passiflora species than the principal component anal- 20)werechosenforeachsample.TheninestudiedPassiflora ysis (data not shown). Biplots of the morphological dataset accessions sequences were compiled and aligned using multi- for discriminant functions (DF) one and two are shown plesequencecomparisonbylog-expectation(MUSCLE)[39]. in Figure 1. The discriminant function analysis that was Other 30 additional in-group sequences were obtained from based on linear combinations of the morphological variables the GenBank database (NCBI) and included in the alignment accounted for 85.69% of the total variance (64.13% in DF1 and (Table 1). Sites where gaps were required to maintain the 21.56% in DF2). All of the morphological characteristics were alignment of the sequences were treated as missing data. loaded heavily on the positive ends of the plot. As in an earlier analysis, Mitostemma brevifilis and Paropsia We produced a scatter plot of 230 specimens for the madagascariensis were chosen as outgroups [32]. first two discriminant functions based on morphological Phylogenetic relationships were developed using maxi- characteristics; the samples were densely arranged, and no mum likelihood (ML) and maximum parsimony (MP) with overlapping characteristics were observed. The discriminant the MEGA 5.1 software40 [ ]. Maximum likelihood is a factors grouped the Passiflora species into five main clusters. method that seeks the tree that makes the data most likely. It The specimens belonging to Group 1 comprised cultivars of applies an explicit criterion—the log—likelihood to compare P. e du li s (PE1, PE2, PE3, and PE4) were highly discriminated the various models of nucleotide substitution in the presence with respect to leaf, stem, sepal, and petal sizes. Accordingly, of a large number of short sequences. Maximum likelihood with the exception of fruit color and fruit sizes, we found tries to infer an evolutionary tree by finding the tree which no significant differences in morphological variables among maximizes the probability of observing the data [41]. The cultivars of P. e du li s .Atripening,P. e du li s (PE1), P. e du li s ML analysis using the Tamura 3-parameter model with (PE2), P. e du li s (PE3), and P. e du li s (PE4) turn purple, dark gamma distributions (T92+G) was selected as the best-fitting purple, pink red, and yellow, respectively. The mean fruit substitution model. The best-fitting substitution method was sizes were also significantly different among cultivars. The chosen based on the Akaike information criterion (AIC), the fruit sampled from Ba’kelalan (PE4) was oval-shaped; conse- Bayesian information criterion (BIC), and ln 𝐿 criterion. The quently, the length of the fruit was statistically comparable to model with the lowest AIC and BIC scores and the highest that of other P. e du li s cultivars that produced round fruits. The ln 𝐿 was chosen to best describe the substitution pattern [41]. fruits of P. e du li s (PE3) (5.92 × 5.57 cm) and P. e du li s (PE2) Likelihood analysis was performed by initially determining (5.48 × 4.68 cm) were smaller than those of P. e du li s (PE1) the transition : transversion ratio (ts : tv) that maximized the (7.95×6.68 cm) and P. e du li s (PE4) (9.02×6.49 cm). Group 2, log-likelihood value; more specifically, the range of ts : tv which was composed of P. incarnata accessions, was related values was plotted against the corresponding inferred log- to the positive ends of the DF1 axis and the negative ends likelihoods. The sequences were analyzed using a heuristic of the DF2 axis. The species of this group were also highly search with bootstrap analysis based on 1000 replicates of discriminated by number of outer corona rows; in addition, the dataset. The resulting were saved and used as this cluster was closely related to the P. e du li s cluster. starting trees for random addition following nearest neighbor Group 3, consisting of P. qu adrang u l ar i s accessions,was intersection (NNI) branch swapping. Maximum parsimony locatedattherightendoftheDF1axis,andthemembers isbasedontheassumptionthatthemostlikelytreeisthe of this group were highly discriminated by flower, fruit, and one that requires the fewest number of changes to explain seed variables. Passiflora quadrangularis produced the largest thenucleotidesequencedatainthealignment.Insteadof flowers with longest coronas of all analyzed species; this The Scientific World Journal 5 b c d b e d c d d b d c b d e b g cd cd (PF2) 1.47 0.14 0.11 1.04 0.02 0.25 0.02 6.19 0.37 0.00 0.26 0.20 0.35 0.04 0.26 0.04 0.04 0.18 0.86 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.38 1.45 1.43 2.05 2.33 1.29 2.35 3.86 6.42 0.29 0.61 0.41 0.25 0.72 0.06 2.00 2.73 7. 2 7 13.38 P. fo eti d a b e d b d d d c c c b e b d c b cd cd fg (PF1) 1.23 0.15 0.01 0.02 0.71 7. 4 5 0.44 0.03 0.03 0.00 0.57 0.47 0.23 0.26 0.89 0.14 0.22 0.03 0.49 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 8.21 1.34 1.38 2.49 1.32 2.16 0.28 7. 3 4 2.32 1.44 0.32 0.77 0.62 4.20 0.06 2.00 16.03 2.73 0.43 a a a b b d f b a d d c b d bc b de cd b 1.15 0.10 0.07 0.05 0.00 0.57 0.32 0.47 0.06 0.10 0.27 0.04 0.05 0.09 0.48 0.75 0.05 0.03 0.66 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± m), PTW-petal width (cm), SPL-sepal length (cm), SPW- accessions. LL-leaf length (cm), LW-leaf width (cm), STW- ), and SW-seed width (cm). 2.1 2.45 1.22 1.28 4.15 8.71 8.37 2.32 6.57 3.42 5.00 4.04 0.42 23.52 0.83 0.07 0.39 2.80 0.56 Passiflora a a a a a b a c a a a a c d a a a a ab 2.53 51.57 0.27 0.70 0.60 0.42 0.11 0.96 0.14 0.12 0.50 0.00 0.01 0.16 0.19 0.06 0.03 0.03 1.08 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± accessions. 4.16 1.85 0.15 4.13 1.22 1.97 2.59 0.98 0.74 6.26 3.00 0.63 12.16 22.38 12.96 13.66 10.42 26.63 2175.0 Passiflora a b a d a b d b a a e b c a e b b a bc species 1.51 0.87 0.49 0.12 0.16 0.14 6.12 0.57 0.05 0.16 0.03 0.85 0.00 0.02 0.02 0.30 0.69 0.03 0.92 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.77 35.5 3.88 1.98 0.14 0.41 6.69 4.06 0.61 4.23 3.79 9.93 0.38 4.30 23.87 2.00 Passiflora 12.03 0.49 10.54 P.maliformis P.quadrangularis P.incarnata P.foetida 0.05) among the means of each variable of the a a b a b b b b c b c b a b a b b bc bc ≤ P Vegetative morphology 0.35 0.52 1.04 (PE4) 4.55 0.51 0.37 0.13 0.48 0.02 0.20 0.02 0.08 0.49 0.26 0.77 0.04 0.38 0.37 Reproductive morphology 0.04 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.55 0.57 1.37 0.13 2.53 9.09 9.02 0.63 0.45 3.46 2.49 3.66 4.00 13.85 1.22 14.67 6.49 124.4 23.56 P. edu li s a a b a b b b b c b b a b ab cd cd cd b bc 0.63 (PE3) 0.92 0.37 0.11 0.01 0.16 0.01 0.05 Table 2: Morphological parameters of nine 0.48 0.09 0.33 0.45 10.91 0.12 0.02 0.23 0.34 0.67 0.42 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.52 0.12 1.37 2.56 2.13 1.34 0.40 0.59 3.63 3.70 13.12 3.30 5.57 8.77 5.92 64.4 14.26 0.58 25.83 P. edu li s a a b a b b b a d b c c c cd ab bc b ab bcd 3.65 0.36 0.91 (PE2) 1.36 0.18 0.02 0.03 0.48 0.09 0.03 0.31 0.58 0.71 0.18 0.07 13.35 0.89 0.04 0.07 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.19 1.15 2.72 9.18 0.14 0.61 4.68 3.57 13.71 3.70 0.40 3.89 1.73 0.56 49.8 5.48 14.08 26.95 2.89 P. edu li s a a b a c b b b b a b b ab bc bc bc b cd bc (PE1) 2.06 0.16 0.69 0.11 0.83 0.03 0.08 0.40 0.08 0.02 0.05 0.02 0.52 1.39 0.54 0.41 14.55 0.07 0.04 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P. edu li s Parameter LWSTWTLTW 14.12 0.59 24.43 BW 0.13 FLSPTLPTW 2.15 SPL 8.91 SPW 3.64 OCR 1.23 CL 3.59 FM 1.27 FL 3.40 FD 2.45 SL 83.3 SW 7.95 6.68 0.63 0.41 BL 3.37 LL 13.82 stem width (cm), TL-tendril length (cm),sepal TW-tendril width width (cm), (cm), ORC-outer corona BL-bract rows, length CL-corona (cm), length BW-bract (cm), width FM-fruit (cm), mass FLS-flower (g), size FL-fruit (cm), length PTL-petal (cm), length FD-fruit (c diameter (cm), SL-seed length (cm Differentsuperscript letters within same the row indicate significant differences (Tukey’s test, 6 The Scientific World Journal

1 2 85.69 1 Variables (axes DF and DF : %)

Bract width Bract length

0.5 Fruit mass Corona length Fruit length Tendril width Fruit width %) Seed width Seed length 0 Flower size 21.56 Stem width Leaf length ( Petal width 2 Leaf width Sepal length DF Sepal width Tendril length

−0.5

Petal length

Outer corona rows −1 −1 −0.5 0 0.5 1 DF1 (64.13%) (a)

1 2 85.69 30 Observations (axes F and F : %)

20 Group 4 Group 5 Group 3 P. malifor mi s 10 ( ) P. foetida PF2 P. qu adrang u l ar i s

%) Group 1 0 ( ) ( ) ( ) 21.56 P. foetida PF1 P. e du li s PE3 P. e du li s PE1 ( ( ) 2 P. e du li s PE4

DF ( ) −10 P. e du li s PE2 Group 2 P. incarnata −20

−30 −30 −20 −10 0 10 20 30 DF1 (64.13%) (b)

Figure 1: (a) Plot of the morphological parameters of the Passiflora accessions. Percentages in parentheses represent the variation in each component. (b) Positions of the DF scores of nine Passiflora accessions relative to DF1 and DF2. species also produced the largest fruit (22.38 × 12.96 cm, maliformis wasclearlyseparatedintoGroup4nearthe with an average weight of 2.0 kg). Simultaneously, the size positive ends of the DF2 axis, and the members of this group of the seed also was statistically different from all other were highly correlated with respect to bract length and width. species assessed. In all species except P. qu adrang u l ar i s ,the The bract structure of P. malifor mi s differed from that of other pulp represented 50–58% of the weight of the fruit; in P. species; in particular, the three bracts of this species fused quadrangularis, the pulp represented 11–15% of the fruit together and formed large cups around the bud or flowers. weight. The exocarp of the fruits of this group was thin, and The bract of P. malifor mi s was twice as large (6.69 ± 0.16 cm) thepulpwasacidic,paleorange,andsweet.Theripened as that of P. e du li s (3.37 ± 0.07 cm). The last group consisted mesocarp was 2.5–3.0 cm thick which is edible. Passiflora of cultivars of P. fo eti d a : P. fo eti d a with green bracts (PF1) The Scientific World Journal 7 and P. fo eti d a with red bracts (PF2). These species clearly rangeofts:tvvaluewasfoundtobe1.515andthisvalue diverged from the other cultivated Passiflora accessions that was used in all subsequent maximum likelihood analyses. were located at the negative ends of the DF1 and DF2 axes. The obtained ML tree was constructed using a heuristic Few differences in plant morphology were observed between search that was performed using the NNI branch swapping these two wild Passiflora cultivars with different bracts and option (− ln 𝐿 = 1426.54). For the MP analysis, the most fruit colors. The plant parts of both species were covered with parsimonious tree generated by phylogenetic analysis had a numerous, small sticky glands and sticky hairs. consistency index (CI) of 0.708, a retention index (RI) of According to Martins et al. [42], the variation observed in 0.805, and a rescaled consistency index (RCI) of 0.569. The fruit morphology is very common even at intraspecific level. bootstrap analyses indicated high support for the main clades The variation may be attributed to environmental factors within the phylogenetic tree. or genetic differences or both. The variations in the fruit The results of algorithms applied (ML and MP) showed traits were attributed to differences in the age of the plant, that samples examined were distributed into two distinct fruit maturation stage, geographical sites, climatic condition, well-supported clades. All of the Passiflora accessions occu- soil properties, and also seed origin [10]. Additionally, the pied separate topological positions; that is, no overlap among complex nondominant inheritance of the external color of species was observed. The resultant MLFigure ( 2(a))and the fruit renders species identification even more challenging, MP trees (Figure 2(b)) were very similar; yet variation in the asanumberofintermediatecolorscanbeproducedinP. bootstrap support values and positioning of certain species edulis [43]. In the present study, the cultivars of P. e du li s were was observed. Subgenus Passiflora was supported as mono- recognized and named accordingly with the International phyletic linkage in both ML and MP trees obtained. This is Code of Botanical Nomenclature based on their significant agreeable with finding of Muschner et al. [32]andKrosnick agronomic characteristics [43]. Assessments of morpholog- et al. [44]. The MP topology was chosen for discussion as ical characteristics grouped the species according to their it showed stronger bootstrap supports and gave insight into similarities. In accordance with the current study, Viana et al. relationship within the subgenus Passiflora with Passiflora [10] assessed the morphological diversity in six wild Passiflora accessions examined arranged following their supersection species and stated the interspecific variability was observed and section or series recognized by Feuillet and MacDougal fornumberofflowers,numberoffruits,seeds,fruitlength, [7]. fruit width, leaf area, and leaf length. Crochemore et al. [9] The MP analysis yielded a most parsimonious tree with obtained clear separation among the 55 accessions consisting high proportion informative sites (37.2%) and resulted in two of 11 species from subgenus Passiflora by using morphological well resolved major clades. As was observed with the ML approach. The authors also recorded high divergence of P. analysis, a similar topological pattern was recorded for the foetida from other Passiflora probably due to the diversity of cultivars of P. fo eti d a in clade 1 (bootstrap score of 99%), species studied and this was agreeable with Viana et al. [16] which was basal to the clade containing other Passiflora who worked with different accessions of cultivated and wild accessions (i.e., P. caerulea, P. incarnata, P. maliformis, P. species of Passiflora. quadrangularis,P.vitifolia,P.alata,P.platyloba,and P. e du li s ). The constructed phylogeny tree was in agreement with the morphological assessment, confirming the divergence of wild 3.2. Sequences Characteristics. The PCR products for all of P. fo eti d a from all other cultivated species. Clade 2 con- the studied species were approximately 680 base pairs in sisted of other Passiflora accessions with moderately strong length. The final dataset of aligned ITS sequence (including bootstrap scores of 87%. In this clade, six well-separated outgroup) used for the phylogenetic analysis consisted of subclades based on species similarities were formed. Subclade 39 accessions from 13 Passiflora species. The final align- 1 consisted of all P. e du li s individuals that clustered as a single ment was highly variable, with 37.2% of sites that were group. The four accessions of P. e du li s from East Malaysia parsimony informative. The guanine-cytosine (GC) content were clustered in the same group and all of these accessions of the Passiflora species ranged from 60% to 64% and were well segregated based on their genetic similarity. In averaged 63%. Muschner et al. [32] reported similar GC agreement with the ML analysis, P. e du li s (PE4) occupied the contents in the Passiflora subgenusandstatedthattheGC base of this subclade. Subclade 2 consisted of P. incarnata content of Passiflora was higher than the GC content (53%) accessions. Subclades 1 and 2 belonged to the Passiflora encountered in other subgenera (i.e., Decaloba, Adopogyne, supersection. , which is a member of the Murucuja, Pseudomurucuja, and Deidamioides). The genetic Coccinea supersection, was clustered in subclade 3 and pairwise distance among taxa estimated with the Tamura 3- formed an independent lineage. Accessions of P. cae r u l e a parameter model was calculated using complete sequence were clustered in subclade 4, with scores of 99%. Passiflora data (including data from outgroups); this distance ranged caerulea was assigned to categories based on morphological from 0 to 45.5% and averaged 16.1%. characteristics belonging to the Stipulata supersection and the Granadillastrum section, as proposed by Feuillet and 3.3. Phylogenetic Analysis. Phylogenetic analyses were per- MacDougal [7]. , which belongs to the formed using maximum likelihood (ML) and maximum StipulatasupersectionandtheDysosmiaseries,evolved parsimony (MP) method. For ML analysis using the Tamura separately from P. cae r u l e a . A similar pattern was recorded 3-parameter model with gamma distributions (T92+G) was by Muschner et al. [32],whoobservedhighdivergence selected as the best-fitting substitution model. The optimal between P. cae r u l e a and P. fo eti d a .Becauseofitsdivergence 8 The Scientific World Journal

1,2,3 EU258379.1 Passiflora edulis (Brazilian state ) 1,2,3 EU258378.1 Passiflora edulis (Brazilian state ) 1,2,3 EU258383.1 Passiflora edulis (Brazilian state ) Passiflora edulis (PE2) ∗ AF454803.1 Passiflora edulis (Netherland ) 1,2,3 EU258382.1 Passiflora edulis (Brazilian state ) 1,2,3 82 69 EU258381.1 Passiflora edulis (Brazilian state ) 1 Passiflora edulis (PE ) 1,2,3 EU258384.1 Passiflora edulis (Brazilian state ) 1,2,3 EU258380.1 Passiflora edulis (Brazilian state ) 1,2,3 EU258376.1 Passiflora edulis (Brazilian state ) Passiflora edulis (PE3) 1,2,3 EU258375.1 Passiflora edulis (Brazilian state ) 2 Passiflora edulis (PE4) AF454796.1 Passiflora vitifolia (unknown) ∗ 85 99 AF454802.1 (Netherland ) EU258315.1 Passiflora caerulea (RS Brazilian state) AF454801.1 (unknown) 454798.1 91 AF Passiflora platyloba (USA) 99 85 AY210956.1 Passiflora maliformis (Dominica) 99 DQ344630.1 Passiflora incarnata (Brazilian state) AY032826.1 (Viamao, RS) Passiflora quadrangularis 65 AF454799.1 Passiflora quadrangularis (FrenchGuiana) AY636107.1 Passiflora quadrangularis (Ohio State) 1,2,4 EU258393.1 Passiflora foetida (Brazilian state ) 1,2,4 EU258389.1 Passiflora foetida (Brazilian state ) DQ521376.1 Passiflora foetida (United States) DQ238783.1 Passiflora foetida (Recife, PE) DQ238784.1 Passiflora palmeri (Brazilian state) 98 1,2,4 1 EU258394.1 Passiflora foetida (Brazilian state ) JQ723359.1 Passiflora foetida (Ecuador) DQ499117.1 Passiflora foetida (tropical regions) 73 Passiflora foetida (PF1) 95 Passiflora foetida (PF2) AY102365.1 Paropsia madagascariensis AY102359.1 Mitostemma brevifilis (a) 1,2,3 84 EU258382.1 Passiflora edulis (Brazilian state ) 258381.1 1,2,3 EU Passiflora edulis (Brazilian state1,2,3) EU258378.1 Passiflora edulis (Brazilian state ) 258383.1 1,2,3 EU Passiflora edulis (Brazilian state∗ ) AF454803.1 Passiflora edulis (Netherland ) Passiflora edulis (PE2) Passiflora 82 Passiflora edulis (PE1) 1,2,3 EU258379.1 Passiflora edulis (Brazilian state ) 71 258384.1 1,2,3 EU Passiflora edulis (Brazilian state1,2,3) Passiflora EU258380.1 Passiflora edulis (Brazilian state ) Passilfora edulis (PE3) 258375.1 1,2,3 EU Passiflora edulis (Brazilian state1,2,3) EU258376.1 Passiflora edulis (Brazilian state ) 2 75 Passiflora edulis (PE4) 99 Passiflora incarnata Passiflora 87 DQ344630.1 Passiflora incarnata (Brazilian state) Coccinea 454796.1 AF Passiflora vitifolia (unknown)∗ 99 AF454802.1 Passiflora caerulea (Netherland ) Granadillastrum EU258315.1 Passiflora caerulea (RS,Brazilian state) 90 AF454801.1 Passiflora ambigua (unknown) Laurifoliae

AF454798.1 Passiflora platyloba (USA) Stipulata 99 AY210956.1 Passiflora maliformis (Dominica) Tiliifolia 95 Passiflora maliformis Passiflora quadrangularis 78 AY636107.1 Passiflora quadrangularis (Ohio State) 68 AF454799.1 Passiflora quadrangularis (French Guiana) Quadrangulares AY032826.1 Passiflora alata (Viamao, RS) Laurifolia 258389.1 1,2,4 EU Passiflora foetida (Brazilian state1,2,4) EU258393.1 Passiflora foetida (Brazilian state ) 1 99 DQ238784.1 Passiflora palmeri (Brazilian state) DQ521376.1 Passiflora foetida (United State) DQ238783.1 Passiflora foetida (Recife, PE) Dysosmia JQ723359.1 Passiflora foetida (Ecuador) 1,2,4 EU258394.1 Passiflora foetida (Brazilian state ) DQ499117.1 Passiflora foetida (tropical regions) Stipulata 74 Passiflora foetida (PF1) 95 Passiflora foetida (PF2) AY102365.1 Paropsia madagascariensis AY102359.1 Mitostemma brevifilis

(b)

Figure 2: (a) Phylogenetic tree of Passiflora accessions inferred from the ML analysis using the Tamura three-parameter model. Only bootstrap scores greater than 60% are shown. Mitostemma brevifilis and Paropsia madagascariensis were used as outgroups. (b) Phylogenetic tree of Passiflora accessions inferred from parsimony analysis (full heuristic search with the tree bisection reconnection method). The Scientific World Journal 9 from other species in the Passiflora subgenus, the placement The level of variation observed in the ITS region dataset of Dysosmia into a separate subgenus, was proposed by was consistent with observations of Muschner et al. [32] Yockteng and Nadot [17] and is supported by the present and Krosnick and Freudenstein [46]andmaybeduetothe finding as evident in Figure 2(b). Subclade 5 comprised percentage of missing sequence data for the four reference P. ambig u a , P. pl at y l ob a , and P. malifor mi s , and subclade 6 sequences used by Muschner et al. [32]; this variation com- was composed of P. qu adrang u l ar i s and P. al ata , which both plicated the final alignment using MUSCLE with different belong to the Laurifolia supersection. The P. malifor mi s and gap extensions. The final alignment was chosen based on P. pl at y l ob a belonged to the same series (Tiliifolia), while P. thecongruenceinpublishedrelationshipsamongoutgroups quadrangularis was placed under series of Quadrangulares. [32, 46]. Besides the ITS region, other markers such RAPD, The present study revealed certain degree of variation AFLP, SSR, and, cpDNA also make a direct comparison detected in the ITS region sequence in all the individuals of these studies difficult and challenging; because of that examined. This is in agreement withader M¨ et al. [31], where intraspecific variation is not evenly distributed among species in Passiflora the ITS region resulted in more informative sites and led to different datasets for the same species (i.e., P. than other markers (i.e., cpDNA). The ITS region provided caerulea, P. e du li s , and P. malifor mi s ). This may be attributed greater resolution at the species level and was useful for to the complexities of the evolutionary history of the genus differentiating the major groups of the Passiflora subgenus. andindicatesthatrobustpatternswouldonlyemergewhen Krosnick et al. [44]alsohavereportedtheITSdataprovide different markers are considered together [31]. greaterresolutionthanncpGSandtrnL-trnFatthespecies The molecular phylogenetic placement of the individuals level within Passiflora.Krosnicketal.[44]alsoshowedthat in Passiflora subgenus were clearly separated than the sub- the subgenus Passiflora is monophyletic (97%) as observed genera of Astrophea or Distephana and was also supported in the present study compared to other subgenera (i.e., by the morphological classification [45]. The present study Deidamioides) which is polyphyletic. showed, the MP phylogenetic tree was congruent with the Pattern of intraspecific variability in Passiflora using ITS classification based on morphological descriptions of Feuillet region has been also studied by Muschner et al. [32]who and MacDougal [7]wherethesubgenusPassiflora are cate- investigate the relationship of 61 species of Passiflora com- gorized into six supersections. Morphologically, supersection posing the entire subgenera which was formally classified in Passiflora comprises those species that have serrate leaves, 11 subgenera and representatives of four other genera. Based free serrate bracts, and upright flowers with a dominating on the phylogenetic tree obtained from ML analysis yielded corona as observed in P. e du li s and P. incarnata.Thesuper- 3 major clades; representing subgenus Passiflora, Decaloba section Stipulata is divided into three sections and section and Astrophea and the position of subgenus Deidamioides Granadillastrum with the richest species comprised plant was undefined. The MP tree obtained was very similar to the with entire 3–5 lobed leaves and conspicuous upright flowers respective ML tree was agrees with the current finding. The with free bracts (i.e., P. cae r u l e a ). Essential characteristics of present results revealed some differences on the positioning the species from supersection Laurifolia are large pendent of few Passiflora species in both the trees. Muschner et al. flowers with predominant corona that surrounds the ovary [32] stated that, although there are some consistent species in a campanulate fashion. This group possessed 3 series. grouping within the Passiflora clade, only few of them Whenthebractsareconnate,atleastatbase,thischaracteris have high support and are consistent among markers and sufficient to assign the species to series Tiliifolia as recorded phylogenetic method, that is, P. qu adrang u l ar i s and P. al ata in P. malifor mi s .TheP. qu adrang u l ar i s is well known species group. Most of this subgroup however, are not consistence of series Quadrangulares because of its winged, angled stems with Killip’s [6] subgenera or sections. andpossesseslarge,unlobedleaveswithentiremargin. In accordance with the present finding, studies by Thus,theclassificationbasedonthemorphologicalchar- Ossowski [45] revealed that within Passiflora,thegroupP. acteristics supports our phylogenetic topology in subgenus menispermifolia, P. oerstedii, and P. cae r u l e a as well as the Passiflora. The present work provides the better integration species pairs P. pl at y l ob a with P. ambig u a and P. qu adrang u - of morphological data and ITS sequences to understand the relationship within subgenus Passiflora. Although our laris with P. al ata are relatively well supported. The authors analyses considered only nine species, our results can be used mentioned that the MP and ML trees obtained were not to study phylogenetic relationships of closely related species congruent and this was in contrast to the ML and MP topolo- and the separate topologies of different species. gies of present study. This contradicts the highly derived position in the parsimony tree. Mader¨ et al. [31]studied the intraspecific genetic diversity in 23 species of genus 4. Conclusion Passiflora consisting of subgenera Decaloba and Passiflora using ITS markers. The work by Mader¨ et al. [31]revealed This study provides an overview of a variety of genetically dif- that the Passiflora and Decaloba subgenera showed significant ferent individuals that could be commercialized in Malaysia differences in the sizes of the ITS regions and in GC content, andusedinfuturebreedingprograms.Ourresultsconfirm which can be related to reproductive characteristics of species that the ITS region provides high resolution at the species in these subgenera. The clear seperation obtained within level and is useful for differentiating the major groups of six species indicated that ITS may be a useful tool for the the Passiflora subgenus. Although the analysis presented here evaluation of intraspecific genetic variation in Passiflora. was based on a limited number of species, the ITS region 10 The Scientific World Journal

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